Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

AbstractOstreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins

Interaction of α-Synuclein with Negatively Charged Lipid Membranes Monitored by Surface Plasmon Resonance

Aggregation of presynaptic protein α-synuclein is implicated in the development of Parkinson’s disease. Interaction of α-synuclein with lipid membranes appears to be critical for its physiological and pathological roles. Anionic lipids trigger conformational transition of α-synuclein from its natively disordered into an α-helical structure. Here we used surface plasmon resonance (SPR) to determine the affinities of α-synuclein for the small unilamellar vesicles composed of anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) or 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. α-Synuclein bound in a concentration dependent manner to equimolar mixtures of POPC/POPS and POPC/DPPG vesicles. The affinity of α-synuclein for POPC/POPS was ~3-fold higher than for POPC/DPPG. These results indicate that headgroup charge is not the only factor contributing to α-synuclein-membrane association. This work is licensed under a Creative Commons Attribution 4.0 International License

EFFECTS OF pH ON HUE ANGLE AND VISIBLE ABSORPTION MAXIMA OF CYANIDIN

As a major sub-group of flavonoids, anthocyanins are water soluble plant pigments responsible for the blue, purple and red color of many plant tissues. They occur primarily as glycosides of their respective aglycone anthocyanidin. Cyanidin is the most abundant anthocyanidin in fruits and vegetables (50%). In recent years, the interest in the properties and stability of anthocyanin extracts has increased. However, there remain little data in the literature relating to the properties and stability of pure anthocyanins, and especially of the anthocyanidins. The aim of the study was to compare under various pH conditions visible absorbance maxima (λmax-vis) with the corresponding hue angle (hab) values of cyanidin as a color reference. Thus cyanidin aqueous solution was subjected to spectroscopic and colorimetric study to examine influence of pH value on hab and λmax-vis. The cyanidin had reddish nuances at the lowest pH values. By stepwise pH increase the colour of cyanidin is gradually changed toward magenta and lilac tones, and then with further pH increase to more reddish nuances. In alkaline region cyanidin showed yellow green tones, which is gradually changed toward yellow nuances with further pH increases.&nbsp

Characterization of ciprofloxacin binding to the linear single- and doublestranded DNA

Abstract The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na + , no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na + , a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g

Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum

Eterična olja origana (Origanum vulgare L.), mete (Mentha piperita L.), janeža (Foeniculum vulgare Mill.), in borovih (Abies alba Mill.) iglic in storžev, ter njihove sestavine timol, karvakrol, mentol in anisaldehid smo testirali kot snovi z protiglivno aktivnostjo proti plesnim vrste Penicillium verrucosum. Najnižje minimalne inhibitorne koncentracije (MICs) smo določili eteričnemu olju origana, ter karvakrolu, timolu, in mentolu. Rast plesni vrste P. verrucosum je bila popolnoma inhibirana po 21 dneh inkubacije v prisotnosti ½ MIC timola (0,0625 mg mL-1), medtem ko so eterično olje origana (½ MIC, ¼ MIC), timol (¼ MIC) in karvakrol (½ MIC, ¼ MIC) inhibirali rast plesni od 8,3 % do 70.8 %. Dodatek mentola v koncentracijah ¼ MIC in ½ MIC (0.1875 mg mL-1 in 0.3750 mg mL-1) pa ni imel inhibitornega vpliva na rast plesni vrste P. verrucosum. Popolno inhibicijo tvorbe ohratoksina A smo določili le v primeru, ko je bila tudi rast teh plesni popolnoma inhibirana (pri ½ MIC timola). Največjo inhibicijo tvorbe ohratoksina A (96,9 %) po 21 dnevih inkubacije smo določili pri ¼ MIC timola (0.0313 mg mL-1). Pospešeno tvorbo ohratoksina A (od 13,9 % do 28,8 %) pa smo določili pri ¼ MIC eteričnega olja origana (0.2930 mg mL-1) in ½ MIC karvakrola (0.1953 mg mL-1). Protiglivni vplivi so bili odvisni od testirane snovi, njene koncentracije ter časa interakcij med protiglivno snovjo in plesnimi vrste P. verrucosum. Rezultati nakazujejo možnosti uporabe eteričnega olja origana v živilih namesto kemijskih konzervansov. Ker pa so živila kompleksna okolja v katerih je potrebno upoštevati več dejavnikov, so potrebne nadaljnje raziskave.Essential oils from oregano (Origanum vulgare L.), mint (Mentha piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones, and their active substances thymol, carvacrol, menthol, and anisaldehyde were tested for antifungal activity against Penicillium verrucosum. The lowest minimal inhibitory concentrations (MICs) were achieved for essential oil of oregano, followed by carvacrol, thymol, and menthol. These antifungal components were further investigated, as the main aim of our study was to elucidate the effect of natural antifungals on ochratoxin A production. During 21 days of exposure, the growth of P. verrucosum, and subsequently the production of ochratoxin A, was fully inhibited by thymol at ½ MIC (0.0625 mg mL-1), but menthol at ¼ and ½ MIC (0.1875 and 3750 mg mL-1) showed no growth inhibition. After 21 days of incubation, the greatest inhibitory effect on ochratoxin production (inhibition was 96.9 %) was also achieved with thymol at ¼ MIC (0.0313 mg mL-1). Essential oil of oregano (¼ MIC, 0.2930 μL mL-1) and carvacrol (½ MIC, 0.1953 μL mL-1) stimulate production of ochratoxin A at 13.9 % to 28.8 %, respectively. The observed antifungal effects depended on the agent, the concentration used, and the time of interaction between the agent and P. verrucosum. Our results indicate the possibility of using oregano essential oil as a substitute for artificial preservatives in certain foods, but further research is needed

Haloarchaeal communities in the crystallizers of two adriatic solar salterns

Abstract: Solar salterns operate only for short dry periods of the year in the north shore of the Adriatic Sea because of its relatively humid and cold Mediterranean climate. In a previous paper, we showed that the NaCl precipitation ponds (crystallizers) of Northern Adriatic Seovlje salterns have different haloarchaeal populations from those typically found in dry and hot climates such as Southern Spain. To check whether there is a common pattern of haloarchaeal diversity in these less extreme conditions, diversity in crystallizers of other Adriatic solar salterns in Ston, Croatia was ascertained by molecular and culture methods. In addition, the cultivation approach was used to further describe haloarchaeal diversity in both salterns. Over the period of two solar salt collection seasons, isolates related to species of the genera Haloferax, Haloarcula, and Haloterrigena were recovered from both salterns. Within the same sampling effort, relatives of the genus Halorubrum and a Natrinema-like isolate were cultivated from Slovenian Seovlje salterns while Halobacterium related isolates were obtained from the Croatian Ston salterns. Concurrent with our previous findings, a library of Croatian saltern crystallizer PCR-amplified 16S rRNA genes was dominated by sequences related to the genus Halorubrum. The microbial community structure was similar in both salterns but diversity indices showed greater values in Slovenian salterns when compared with Croatian salterns. Key words: 16S rRNA, Haloarchaea, saltern, hypersaline, halophiles. Résumé : Les salines solaires ne fonctionnent que pendant de brèves périodes de temps sec dans l'année sur la rive nord de la mer Adriatique à cause de son climat méditerranéen relativement humide et froid. Dans un article précédent, nous avons démontré que les bassins de précipitation de NaCl (cristalliseurs) des salines de Seovlje de l'Adriatique Nord renferment des populations haloarchéenes différentes de celles normalement retrouvées dans des climats chauds et secs tels que le sud de l'Espagne. Pour vérifier s'il existe une configuration commune à la diversité haloarchéene dans ces conditions moins extrêmes, nous avons décrit la diversité dans les cristalliseurs d'autres salines solaires adriatiquesles salines Ston en Croatie -par des méthodes moléculaires et de culture. De plus, l'approche de culture fut employée afin de décrire davantage la diversité haloarchéene des deux salines. Sur une période de deux saisons de collecte de sel solaire, des isolats apparentés à des espèces des genres Haloferax, Haloarcula et Haloterrigena ont été recueillis des deux salines. À l'issue d'un même échantillonnage, des parents du genre Halorubrum et un isolat semblable à Natrinema ont été cultivés à partir de la saline slovène de Seovlje alors que des isolats apparentés à Halobacterium ont été obtenus des salines Croates de Ston. En conformité avec nos découvertes précédentes, la banque de gènes de ARNr 16S amplifiés par PCR du cristalliseur salin Croate était dominée par des séquences apparentées au genre Halorubrum. La structure de la communauté microbienne était semblable dans les deux salines mais les indices de diversité ont démontré des valeurs supérieures dans les salines slovènes comparativement aux salines croates

Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)

This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β2-adrenergic receptor (β2-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR. © 2019, The Author(s)

‐sitosterol and gentisic acid loaded 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine liposomal particles

The aim of the present study was the examination of the impact of -sitosterol andgentisic acid on the characteristics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) liposomal particles: (a) bilayer permeability (fluorescence spectroscopy),(b) particle size, polydispersity index (PDI) and zeta potential (photon correlationspectroscopy) and (c) thermal properties (differential scanning calorimetry). -sitosterol induced the increase of liposomal bilayer rigidity, due to rearranging ofthe phospholipid chains, while gentisic acid enhanced the membrane fluidity, dueto the reduced orderliness and the increase of phospholipid dynamics. The inclusionof -sitosterol in liposomes caused a significant increase in particle diameterand PDI, while the encapsulation of gentisic acid did not have influence on particlesize distribution. Apart from that, the presence of -sitosterol resulted in thesignificant zeta potential increase, and thus a better stability of liposomal spheres(in the absence and in the presence of gentisic acid). -sitosterol decreased maintransition temperature (Tm) and phase transition enthalpy (H), and caused thedisappearance of the pre-transition peak as well, whereas the presence of gentisicacid produced a slight decrease in Tm and increase of H. Therefore, gentisic acidhad more favourable, stabilizing interactions with phospholipids than -sitosterol.Thus, it can be concluded that -sitosterol is located in the bilayer interior betweenphospholipids acyl chains, and gentisic acid is incorporated near the outer leaflet ofthe phospholipid membrane, next to the polar head groups. -sitosterol and gentisicacid loaded DPPC liposomal particles have a potential to be used in food andpharmaceutical products, due to the important individual and possible synergisticbeneficial health properties of -sitosterol and gentisic acid

Fluorimetrijska analiza permeabilnosti lipozomalnih membrana

The present study provides insight into changes in the liposomal membrane fluidity as a result of variation of structural components (two types of standard phospholipids - 1,2-dipalmitoyl-sn-glycerero3-phosphocholine, i.e. DPPC and 1-palmitoyl-2-oleoylglycerol-3-phosphocholine, i.e. POPC, one type of phospholipid mixture - Lipoid H and sterols - cholesterol and b-sitosterol), as well as variation in sterol concentration (5-50%). Fluorescence spectroscopy (anisotropy, r) has shown that DPPC liposomal membranes were more rigid compared to POPC and Lipoid H lipid bilayers. Furthermore, the increment of sterol concentration has caused a decrease in the membrane fluidity in all liposomal samples. On the other hand, phospholipid bilayers containing b-sitosterol has possessed better fluidity in comparison to their cholesterol parallels. Higher permeability of b-sitosterol liposomal membranes, as well as the beneficial effects of phytosterol on human health, favor the use of b-sitosterol in the preparation of liposomes as components of food, pharmaceutical and cosmetic products.Prikazana studija daje uvid u promene fluidnosti lipozomalne membrane koje nastaju kao posledica promene strukturnih komponenata lipozoma (dve vrste standardnih fosfolipida - 1,2-dipalmitoil-snglicero-3-fosfoholin, odnosno DPPC i 1-palmitoil-2-oleoil-sn-glicero-3-fosfoholin, odnosno POPC, jedne vrste komercijalne fosfolipidne smeše - Lipoid H i sterola - holesterol i b-sitosterol), kao i promene udela sterola (0-50 mol %). Fluorimetrijska spektrofotometrija (anizotropija, r) pokazala je da su DPPC lipozomalne membrane bile rigidnije u poređenju sa POPC i Lipoid H lipidnim dvoslojima. Pored toga, povećenje koncentracije sterola prouzrokovalo je smanjenje permeabilnosti membrane u svim lipozomima. Sa druge strane, fosfolipidni dvosloji koji su sadržali b-sitosterol pokazali su veću fluidnost u poređenju sa njihovim paralelama sa holesterolom. Bolja permeabilnost b-sitosterol lipozomalnih membrana, kao i blagotvorni uticaji fitosterola na zdravlje ljudi favorizuju primenu b-sitosterola u pripremi lipozoma kao sastavnih delova prehrambenih, farmaceutskih i kozmetičkih proizvoda